This invention relates to a method for imaging and harvesting cells from a microbial colony on a thin film culture device.
Many recombinant and molecular cloning techniques rely on the ability to culture bacteria on an agar plate and to select particular colonies from the agar for further study. Each colony is typically selected manually with a sterile toothpick, which can be quite laborious. In addition, there can be uncertainty when a researcher attempts to relocate the same colony from the original agar plate.
Accordingly, automated systems have been used to identify and mark a colony growing on a culture device. For example, automated colony picking systems, such as a BIO-PICK automated colony picking system sold by Biorobotics, Inc., Cambridge, U.K., have been developed to increase the speed with which recombinant E. coli colonies can be processed for genetic research. Typically, these systems include an imaging component, such as a CCD camera, and a robotic arm that positions a xe2x80x9cpinxe2x80x9d over each colony and mechanically xe2x80x9cpicksxe2x80x9d a portion of the colony material from agar culture plates. The colony material from the agar plates is then transferred to culture medium or reagents for growth of the cells or for amplification or analysis of the genetic material within the transferred material.
The invention features thin film culture devices with positioning structures and methods for harvesting cells from colonies present on such culture devices. Images of the culture devices are obtained and positions of colonies growing or present on such culture devices are identified relative to the positioning structures to allow cells to be harvested from colonies based on the identified positions of the colonies. The positioning structures are useful for realigning the culture device such that cells from colonies on the culture device can be harvested at any time.
In one embodiment, the invention is a culture device for the propagation or storage of microorganisms. The device includes a self-supporting, waterproof substrate and a cover sheet (e.g., a transparent cover sheet), wherein a gelling agent is contained on the self-supporting substrate, and wherein the self-supporting substrate and the cover sheet include positioning structures, e.g., holes, slits, slots, beveled edges, notches, or raised structures. The culture device may further include a barcode label on a surface of the culture device. The self-supporting substrate may further include a spacer and/or a growth medium (e.g., containing one or more nutrients). The culture device may further include an indicator and a corresponding inducer. The cover sheet may further include a gelling agent and/or a reinforcement layer, such as a foam, a film, or a non-woven material.
In another embodiment, the invention is a culture device for the propagation or storage of microorganisms that includes first and second layers that are separable from each other. The first and second layers may include a gelling agent such as guar gum, xanthan gum, locus bean gum, polyvinyl alcohol, carboxymethylcellulose, alginate, polyvinylpyrrolidone, gellan, or low monomer content polyacrylic acid. The first and second layers also include positioning structures such as holes, beveled edges, slits, slots, notches, or raised structures. The first or second layers may include a spacer. The first layer may further include a growth medium. The growth medium may include a detergent or a salt. The first layer may also include a selectable agent. The first or second layer may further include a reinforcement layer.
In another embodiment, the invention is a system for harvesting cells from a colony on a thin film culture device having positioning structures. The system includes a scanner, a processing unit and a picking apparatus. The scanner obtains and provides an image file to the processing unit. The processing unit identifies and selects, if necessary, cell colonies on the culture device and provides the position of the colonies relative to the positioning structures to the picking apparatus. The picking apparatus harvests the cells from the colonies based on the position. The picking apparatus may have an orienting unit, wherein the orienting unit has receiving structures adapted to receive corresponding positioning structures in the culture device. The orienting unit may further include a compliant pad. The picking apparatus can include a liquid handling tip.
In yet another embodiment, the invention is a picking apparatus for harvesting cells from a colony on a thin film culture device having positioning structures. The picking apparatus includes an orienting unit, wherein the orienting unit positions the colony relative to the positioning structures; and a picking arm, wherein the picking arm is programmed with the position of a selected colony relative to the positioning structures and is adapted to contact cells of the selected colony based on the position. The orienting unit has receiving structures adapted to receive corresponding positioning structures in the culture device.
A method for harvesting cells from colonies on a culture device also is another embodiment of the invention. The method includes the steps of providing a thin film culture device having positioning structures; obtaining an image of the culture device including cell colonies on the surface of the device (e.g., by scanning the culture device); processing the image to provide positions of cell colonies relative to the positioning structures of the device; optionally selecting particular cell colonies; and then contacting the cell colonies with a picking apparatus based on the position and/or selection of cell colonies to harvest the cells. The picking apparatus may be moved in at least one or at least two directions from the contact point to harvest the cells. Processing the image may include identifying a location of the positioning structures; identifying a location of one or more colonies, optionally selecting a specific colony; and calculating a position of the selected colony relative to the positioning structures. The position of the colonies relative to the positioning structure may include X-Y coordinates.
In another embodiment, the invention is a computer readable medium having instructions thereon causing a programmable processor to display an image of a thin film culture device having positioning structures on a display device; differentiate positioning structures from colonies on the culture device; identify locations of the positioning structures; identify locations of the colonies and/or selected colonies; and calculate positions of the colonies relative to the positioning structures. The computer readable medium may be a storage medium for storing instructions or may be a transmission medium for transmitting the instructions.
The invention includes a computer readable medium having an image stored therein, wherein the image contains image data representative of colonies on a thin film culture device having positioning structures and a computer readable medium having data stored therein, wherein the data are the coordinates of colonies on a culture device relative to positioning structures on the culture device.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.